scholarly journals A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner

2015 ◽  
Vol 26 (10) ◽  
pp. 1957-1970 ◽  
Author(s):  
Kentaro Umeda ◽  
Nariaki Iwasawa ◽  
Manabu Negishi ◽  
Izumi Oinuma
Keyword(s):  
Neuronal Development ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Binding Domain ◽  
Dominant Negative ◽  
Actin Binding ◽  
Axon Branching ◽  
Axonal Development ◽  
Actin Binding Domain ◽  
Ras Family

Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin–binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms—l-afadin, having the F-actin–binding domain, and s-afadin, lacking the F-actin–binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin–binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras–mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin–regulated axon branching.

scholarly journals R-Ras controls axon branching through afadin in cortical neurons

2012 ◽  
Vol 23 (14) ◽  
pp. 2793-2804 ◽  
Author(s):  
Nariaki Iwasawa ◽  
Manabu Negishi ◽  
Izumi Oinuma
Keyword(s):  
Cortical Neurons ◽  
Ectopic Expression ◽  
Axon Growth ◽  
Small Gtpase ◽  
Microtubule Assembly ◽  
Actin Binding ◽  
Axon Branching ◽  
Branch Number ◽  
Growth Guidance ◽  
Neurite Development

Regulation of axon growth, guidance, and branching is essential for constructing a correct neuronal network. R-Ras, a Ras-family small GTPase, has essential roles in axon formation and guidance. During axon formation, R-Ras activates a series of phosphatidylinositol 3-kinase signaling, inducing activation of a microtubule-assembly promoter—collapsin response mediator protein-2. However, signaling molecules linking R-Ras to actin cytoskeleton–regulating axonal morphology remain obscure. Here we identify afadin, an actin-binding protein harboring Ras association (RA) domains, as an effector of R-Ras inducing axon branching through F-actin reorganization. We observe endogenous interaction of afadin with R-Ras in cortical neurons during the stage of axonal development. Ectopic expression of afadin increases axon branch number, and the RA domains and the carboxyl-terminal F-actin binding domain are required for this action. RNA interference knockdown experiments reveal that knockdown of endogenous afadin suppressed both basal and R-Ras–mediated axon branching in cultured cortical neurons. Subcellular localization analysis shows that active R-Ras–induced translocation of afadin and its RA domains is responsible for afadin localizing to the membrane and inducing neurite development in Neuro2a cells. Overall, our findings demonstrate a novel signaling pathway downstream of R-Ras that controls axon branching.

scholarly journals The Actin-Binding Domain of Slac2-a/Melanophilin Is Required for Melanosome Distribution in Melanocytes

2003 ◽  
Vol 23 (15) ◽  
pp. 5245-5255 ◽  
Author(s):  
Taruho S. Kuroda ◽  
Hiroyoshi Ariga ◽  
Mitsunori Fukuda
Keyword(s):  
Amino Acids ◽  
Binding Domain ◽  
Dominant Negative ◽  
Actin Binding ◽  
Dominant Negative Effect ◽  
C Terminus ◽  
Defective Mutant ◽  
Actin Binding Domain ◽  
Negative Effect ◽  
Myosin Va

ABSTRACT Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (ΔABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

scholarly journals F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain

2016 ◽  
Vol 310 (1) ◽  
pp. C89-C98 ◽  
Author(s):  
Yongtong Zhao ◽  
Sandor S. Shapiro ◽  
Masumi Eto
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Skeletal Development ◽  
Myosin Ii ◽  
Ectopic Expression ◽  
Focal Adhesions ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Filamin B

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants W148R and E227K than the wild-type protein to the cytoskeleton. Ectopic expression of FLNB-W148R and, to a lesser extent, FLNB-E227K induced prominent F-actin accumulations and the consequent rearrangement of focal adhesions, myosin II, and septin filaments and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal rearrangement was partially attenuated by the inhibition of myosin II, p21-activated protein kinase, or Rho-associated protein kinase. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization, preventing normal skeletal development.

scholarly journals A mutation in the LMOD1 actin-binding domain segregating with disease in a large British family with thoracic aortic aneurysms and dissections

2017 ◽  
Author(s):  
Y.B.A. Wan ◽  
M.A. Simpson ◽  
J.A. Aragon-Martin ◽  
D.P.S. Osborn ◽  
E. Regalado ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Binding Domain ◽  
Dominant Negative ◽  
Aortic Aneurysms ◽  
Actin Binding ◽  
Dominant Negative Effect ◽  
Consecutive Series ◽  
Loss Of Function ◽  
Thoracic Aortic Aneurysms ◽  
Actin Binding Domain

AbstractWe describe a mutation in LMOD1, which predisposes individuals to thoracic aortic aneurysms and dissections in a large multi-generation British family. Exome variant profiles for the proband and two distantly related affected relatives were generated and a rare protein-altering, heterozygous variant was identified, present in all the exome-sequenced affected individuals. The allele c.1784T>C, p.(V595A) in LMOD1 is located in a known actin-binding WH2 domain and is carried by all living affected individuals in the family. LMOD1 was further assessed in a consecutive series of 98 UK TAAD patients and one further mutation was found, yielding an incidence of ∼2% in our study group. Assessment of LMOD1 in international TAAD cohorts discovered nine other missense variants of which three were classed as likely pathogenic.Validation of LMOD1 was undertaken using a zebrafish animal model. Knock-down of both lmod1a and lmod1b paralogs using morpholino oligonucleotides showed a reproducible abnormal phenotype involving the aortic arches under off-target controls. Injection of the human LMOD1 c.1784T>C, p.(V595A) mutation demonstrated a likely dominant negative effect and illustrated a loss of function cause.Mutations found in the WH2 actin-binding domain of LMOD1 may delay actin polymerization and therefore compromise actin length, dynamics and interaction with myosin in the smooth muscle contraction pathway.

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

2006 ◽  
Vol 17 (11) ◽  
pp. 4720-4735 ◽  
Author(s):  
Alistair N. Hume ◽  
Abul K. Tarafder ◽  
José S. Ramalho ◽  
Elena V. Sviderskaya ◽  
Miguel C. Seabra
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Actin Binding ◽  
Binding Studies ◽  
Null Cell ◽  
Coiled Coil Region ◽  
Actin Binding Domain ◽  
Transport Assay ◽  
Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

2013 ◽  
Vol 24 (23) ◽  
pp. 3710-3720 ◽  
Author(s):  
Scott D. Hansen ◽  
Adam V. Kwiatkowski ◽  
Chung-Yueh Ouyang ◽  
HongJun Liu ◽  
Sabine Pokutta ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Structure ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Underlying Mechanisms ◽  
Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

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